Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of functions. It was first described as interferon-β2 plasmacytoma growth factor, and hepatocyte stimulating factor. Later on it was described as human B-cell-stimulating factor (BSF2). In 1988 it was proposed to name it IL-6 as further studies have demonstrated that the protein also shows activities not only on B-cells but also on T-cells, hematopoietic stem cells, hepatocytes and brain cells.
IL-6 is produced from a single gene encoding a product of 212 amino acid peptide with a molecular weight between 22-27 kDA. Further, it was reported that also immunoreactive complexes in the range of 60-70 kDA were detected in human body fluids in patients with acute bacterial infections.
IL-6 belongs to the cytokine-family. Cytokines are small molecules secreted from one cell that signals to other cells by binding to its specific receptor. An interleukin is generally a cytokine produced by leucocytes. Pro-inflammatory cytokines (IL-1, IL-6) are predominantly produced by activated immune cells involved in the amplification of inflammatory reactions. Anti-inflammatory cytokines (IL-1receptor antagonist, IL-4, IL-6, IL-10, IL-11, IL-13) act in concert with specific cytokine inhibitors and soluble cytokine receptors to control the pro-inflammatory cytokine response. IL-6 plays a crucial role as well by being the chief stimulator of acute-phase proteins, e.g., CRP, as well as by controlling the level of proinflammatory response. In homeostasic conditions IL-6 concentrations are low whereas under stress conditions amounts of IL-6 increase quickly.
IL-6 production is rapidly induced in the course of acute inflammatory reactions associated with injury, trauma, stress, infection, brain, death, neoplasia, and other situations.
The quantitative determination of IL-6 in serum and plasma may be performed by using immunoassays. The lack of stability of IL-6 protein in the liquid phase is a major disadvantage of the current IL-6 detection assays, and the available methods for the stabilization of proteins are not suitable to stabilize IL-6 (Chamani, A. A. et al., J. Colloid Interface Sci. 297 (2006) 561).
Protein stabilization methods can be divided into two classes. First, stabilization is performed to prevent or minimize chemical modifications of the protein, e.g., protection against oxidation reactions. Chemical modifications are based on changes regarding covalent atom-bonds. More precisely, chemical modifications encompass deamidation reactions, oxidations, hydrolysis and cleavage or formation of new disulfidbridges. Stabilization against chemical modifications of a given protein is often achieved by changes in pH and buffer composition. Formation of new disulfidbridges can be prevented by use of specific protection groups for thiols. Functional groups sensitive towards oxidation can be protected by use of scavenger reagents. Protection is achieved by preferential oxidation of the scavenger.
Secondly, a given protein can be stabilized to prevent physical modifications of the protein, e.g., protection of the protein against conformational changes in the protein structure. Different strategies may be used to prevent these conformational changes. Timasheff S. N., Control of protein stability and reactions by weakly interacting cosolvents. Adv. Protein Chem. 91 (1998) 355-432, describe the addition of certain excipients such as sugars, salts, polyalcohols to stabilize protein in solution. When present in high concentration, these excipients protect against deterioration of the activity and maintain proteins in a functional state.
However, Kendrick B. S., Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: Role in restricted conformational mobility and compaction of native state. PNAS USA 94 (1997) 11917-11922, disclose that, in the presence of sucrose, increases in protein surface area of the interleukin 1 receptor antagonist are more thermodynamically unfavorable than in water. The equilibrium between states is shifted towards that with the smallest surface area. Jensen W. A., Stability studies on maize leaf phosphoenoloyruvate carboxylase: The effect of salts. Biochemistry 34 (1995) 472-480, describes the effect of high concentrations of several salts on the stability of PEPC in solution. Tsai P. K. Use of specific ligands, e.g. use of Heparin for stabilization of aFGF Formulation design of acidic fibroplast growth factor. Pharm Res 10 (1993) 649-659 found that a wide variety of polyanions stabilize acidic fibroblast growth factor (aFGF) by raising the temperature at which the protein unfolds. Campell P J. Et al., II. Procedures used for the production of biological standards and reference preparations. J Biol Stand 2 (1974) 259-267, describe methods for achieving stable and reliable biological standards and reference preparations. The effect of added carbohydrates on the chemical composition and antigenic activity of, e.g., bovine serum albumin is reported in Tarelli E, et al., Additives to biological substances. I. Effect of added carbohydrates on bovine serum albumin. J Biol Stand; 9 (1981) 121±130. Furthermore, Tarelli E, et al., Recombinant human albumin as a stabilizer for biological materials and for the preparation of international reference reagents. Biologicals [1045-1056] (1998) vol: 26 iss: 4 pg: 331, show that recombinant human albumin can be used as a stabilizer for biological materials and for the preparation of international reference reagents. Todd M. J. et al., Dimerization of sensitive proteins the structural stability of the HIV-1 protease. J Mol boil 283 (1998) 475-488, describes that dimerization of sensitive proteins, e.g., HIV-1 protease leads to the structural stability of the protein. Finally, Lougheed W. D. et al., Use of neutral surfactants: Physical stability of insulin formulations. Diabetes (1983) 34:424, describes that the aggregation of insulin into high-molecular-weight polymers may be inhibited by reducing the effective polarity of the solvent.
However, the hitherto known method for stabilizing proteins suffer from the disadvantage that they do not allow sufficient stabilization of IL-6 and in particular of IL-6 in serum.
Consequences of the reduced IL-6 stability are difficulties during the production of IL-6 based reagents, such as restrictions in maximum process times, reduced lot sizes due to the available short process time and limited half life of IL-6. To compensate for IL-6 loss during process time, additional IL-6 has to be added during the inprocess-control which results in increased cost. On the customer side, the reduced stability leads to inconveniently short stability of IL-6 products, e.g., calibrators or quality control materials.
EP 1882944 describes tetradecyl trimethylammonium bromide (TTAB) as a denaturing agent for demasking the epitopes responsible for antibody binding on amyloid-peptide oligomers by removing attached proteins.
US 2004/0157218 discloses a method of treating a biological sample for extraction of nucleic acid, dodecyl trimethylammonium bromide (DTAB) and cethyl trimethylammonium bromide (CTAB) are utilized as the detergent for denaturation of the protein. WO 2009/048962 discloses a separation medium for capillary electrophoretic size separation of proteins in the presence of a compound.
EP 1566437 discloses a method for adsorbing a nucleic acid from a biological sample to a solid phase. CTAB is disclosed as a detergent in the lysis buffer for protein denaturation. Thus, according to the above documents CTAB and DTAB are used to destabilize proteins rather than stabilizing them.
EP 1242576 discloses an aqueous reagent composition to enhance the stability of antigens. The disclosed reagent composition comprises a buffer, a protein-rich blocking agent comprising a mixture of protein and/or polypeptides and having a total protein concentration of from 1 to 50 g %, a solubilizing agent, a salt, a chelating agent, a detergent, and a preservative and having a final pH of 7.5 to 8.5. The preservative may be compound trimethyltetradecylammonium bromide (TTAB) in a final concentration of 0.01% w/v, preferably between 1% and 0.1%. Furthermore, the protein rich blocking agent may be fetal calf serum in a concentration of (5%) w/v. Notably, the preservative TTAB is used to prevent growth of microorganism and not to stabilize antigens.
There is experimental evidence showing that most part of the concentration range for TTAB and the low serum concentration disclosed in EP 1242576 does not stabilize IL-6. On the contrary, under these conditions TTAB even leads to an increased destabilization of IL-6.
A need for a method and means to increase the stabilization of IL-6 still remains. It is therefore the aim of the present invention to find conditions which lead to an improved stability of IL-6 used for quality control material, calibrators, receptor based assays or other assays.